The fertilization process is a critical step in plant reproduction. However, the mechanism of action and mode of regulation of the fertilization process in gymnosperms remain unclear. In this study, we investigated the molecular regulatory networks involved in the fertilization process in Korean pine ovules through anatomical observation, physiological and biochemical assays, and transcriptome sequencing technology. The morphological and physiological results indicated that fertilization proceeds through the demise of the proteinaceous vacuole, egg cell division, and pollen tube elongation. Auxin, cytokinin, soluble sugar, and soluble starch contents begin to decline upon fertilization. Transcriptomic data analysis revealed a large number of differentially expressed genes at different times before and after fertilization. These genes were primarily involved in pathways associated with plant hormone signal transduction, protein processing in the endoplasmic reticulum, fructose metabolism, and mannose metabolism. The expression levels of several key genes were further confirmed by qRT-PCR. These findings represent an important step towards understanding the mechanisms underlying morphological changes in the Korean pine ovule during fertilization, and the physiological and transcriptional analyses lay a foundation for in-depth studies of the molecular regulatory network of the Korean pine fertilization process.

In order to simplify the experimental procedure and treatment procedure, we preserved the embryonic callus (EC) of Fraxinus mandshurica more efficiently. In this paper, we established a method for cryopreservation of EC of F. mandshurica by vitrification. EC was subcultured for 7–10 days (d). Vigorous EC with good growth conditions were selected, and cryopreservation was performed by vitrification. The best pre-culture method was to pre-culture EC on 0.5 mol·L−1 sucrose medium for 3 d, load and culture in the liquid woody plant medium (WPM) supplemented with 2 mol·L−1 glycerol and 0.4 mol·L−1 sucrose for 60 min, then dehydrate in 2 mL of plant vitrification solution 2 (PVS2) (30% glycerol + 15% dimethyl sulfoxide (DMSO) + 15% ethylene glycol + 0.4 mol·L−1 sucrose + liquid WPM). EC was rewarmed in a 40 °C water bath for 2 min after cooling in liquid nitrogen. The procedure for cryopreservation of F. mandshurica EC by the vitrification method established in this experiment is relatively reliable. The results from the present study provide a technical reference for improving the cryopreservation of F. mandshurica EC.